The polypeptide antibiotic gramicidin A and the polyene antibiotic amphotericin B are the best characterized macromolecules which form multimeric ion-specific transmembrane channels. Very recently evidence from chemical labeling, selective extraction, and reconstitution experiments have suggested that the red cell membrane protein Band 3, and possibly also glycophorin A, may play some role in regulating the anion permeability of the human red blood cell. We propose to use spectroscopic techniques to study the membrane structure of these antibiotics and human red cell proteins. Our long term goal is to determine how the chemical structure of the macromolecule in its membrane environment determines the structure of the channel and its function in selectively facilitating the diffusion of ions. We have already used fluorescence techniques to demonstrate that all of the gramicidin molecules in liposomes are involved in dimer channels; thus, spectroscopic measurements on gramicidin in lipsomes will characterize the channel. We seek to determine the state of aggregation, conformation, orientation and mobility of antibiotic molecules in the channels. We plan to incorporate specific fluorescent labels, spin-labels, and 13C nuclei in addition to taking advantage of the intrinsic chromphores already present in these molecules. Fluorescence energy transfer, linear, and circular dichroism, decay of fluorescence anisotropy, and 13C nuclear magnetic resonance relaxation measurements will be carried out. Fluorescence energy transfer will be used to study the aggregation of fluorescently labeled derivatives of the red cell membrane protein glycophorin A in detergents and in reconstituted membranes. This technique will also be used to look for complex formation between glycophorin A and Band 3 in reconstituted membranes. These studies may provide insight into the structural basis for red cell anion permeability and the intramembrane particles seen by freeze-fracture electron microscopy.